This disclosure references various publications, patents and published patent specifications by an identifying citation. The full citations for the disclosures are found immediately preceding the claims. The disclosures of these publications, patents and published patent specifications and all referenced documents are hereby incorporated by reference into the present disclosure in their entirety to more fully describe the state of the art to which this invention pertains.
The basic components that comprise the canonical nucleosome core particle are evolutionarily conserved in all eukaryotes. Within a living cell, however, there exists a remarkable degree of heterogeneity in nucleosome composition that influences chromatin structure and function (Luger et al. 2012). This is exemplified by the various covalent posttranslational modifications (PTMs) of the N-terminal tails (NT) of the core histone proteins (H3, H4, H2A, H2B) that alter nucleosome composition to regulate fundamental DNA-templated programs such as transcription (Zentner and Henikoff 2013). For example, acetylation of the H3NT can directly facilitate transcription by destabilizing nucleosome structure whereas methylation of the H3NT can indirectly regulate transcription by binding effector proteins that stimulate or repress transcription (Bannister et al. 2001; Shogren-Knaak et al. 2006). Recent landmark reports determined that differences in the “epigenomic signatures” of several H3NT PTMs are strongly correlated to the cell type-specific gene expression programs observed in >250 normal and diseased human tissues (Polak et al. 2015; Roadmap Epigenomics et al. 2015). These studies demonstrate that precise alterations of the epigenome are essential in the regulation of gene pathways necessary for the derivation of normal and aberrant cell types.
Altering epigenomic signatures by “erasing” H3NT PTMs can be achieved by several different enzymatic mechanisms. One mechanism is the selective removal of specific H3 PTMs by histone-modifying enzymes such as deacetylases and demethylases (Black et al. 2012; Seto and Yoshida 2014). A more extreme mechanism involves ATP-dependent deposition of a new H3 resulting in the removal of all pre-existing PTMs and associated interacting proteins (Narlikar et al. 2013). An alternative intermediate mechanism between the specific and complete erasure of H3 PTMs is proteolysis of the H3NT, which selectively removes pre-existing H3NT PTMs and associated interacting proteins without affecting the H3 core region (Azad and Tomar 2014; Dhaenens et al. 2015). While retention of the H3 core region preserves nucleosome structure, lack of the H3NT destabilizes intra- and inter-nucleosomal interactions that may increase DNA accessibility and facilitate factor binding (Allan et al. 1982; Andresen et al. 2013; Nurse et al. 2013). Therefore, H3NT cleavage provides an efficient means to rapidly and drastically alter chromatin structure and function both directly and indirectly.
Although proteolysis of histones within chromatin was first reported over 55 years ago, the mechanisms and biological functions of histone cleavage remain largely unknown (Phillips and Johns 1959). Proteolysis of the H3NT has been detected in various single and multi-cellular eukaryotes indicating that H3NT cleavage is an evolutionarily conserved process that likely functions in epigenetic regulation (Allis et al. 1980; Bortvin and Winston 1996; Duncan et al. 2008; Pauli et al. 2010; Duarte et al. 2014; Vossaert et al. 2014). Consistent with this, H3NT cleavage is frequently observed during mammalian developmental programs including embryonic stem cell differentiation, mammary gland development and myogenesis (Duncan et al. 2008; Asp et al. 2011; Khalkhali-Ellis et al. 2014; Vossaert et al. 2014). The recent identification of Cathepsin L and D as the principal H3NT protease during mouse embryonic stem cell differentiation and mammary gland development, respectively, suggests that precursor cells utilize different H3NT proteases in a differentiation-dependent context (Duncan et al. 2008; Khalkhali-Ellis et al. 2014). While these reports imply that targeted H3NT proteolysis at specific genomic regions facilitates differentiation, the lack of a method to identify H3NT-cleaved regions has precluded significant insights into the mechanistic functions of H3NT proteolysis. Thus, a need exists in the art and this disclosure satisfies this need and provides related advantages as well.